Metal ions, such as radionuclides and transition metals, are useful for many diagnostic and therapeutic techniques. For example, metal ions can be used as diagnostic or therapeutic agents, particularly with regard to radioimmunodetection, radioimmunotherapy, magnetic resonance imaging, photodynamic therapy or other similar modalities. However, these techniques require specific targeting of the metal ion to a selected tissue. Targeting molecules (such as antibodies or binding molecules) and/or targetable molecules (such as haptenic peptides) can be used to target metal ions to selected tissues, where the targeting molecule or targetable molecule is conjugated to a chelator. Antibody-chelator conjugates (or peptide-chelator conjugates) are useful because the chelator moiety of these conjugates can bind metal ions to form a metal chelate.
Antibody-chelator conjugates can be used to directly target the metal chelate to a targeted tissue., whereas peptide-chelator conjugates are typically used in combination with a bi-specific binding molecule. For example, the peptide-chelator conjugates may include a hapten that is recognized by a bi-specific binding molecule that also recognizes the targeted tissue. As such, the bi-specific binding molecule can be used to localize the peptide-chelator conjugate (i.e., the targetable molecule) to the targeted tissue.
The stability of these conjugates is important, because if the chelator becomes disassociated from the antibody or peptide, the free chelator can compete with the conjugate for binding to the diagnostic or therapeutic metal ion. Ultimately, this results in a lower percentage incorporation of the metal ion into the antibody- or peptide-chelator conjugate.
As such, there is an ongoing need to prepare stable conjugates of chelating agents and metal chelates with proteins, polymers, polypeptides and peptides, such that the synthesized conjugates remain useful for high incorporation of metals, over an extended period of time. One must ensure that the chelating agent is able to stably and reproducibly bind to a metal ion of interest, and continue to do so in a high incorporation yield, over an extended period of time after manufacture of the chelator-protein conjugate. By incorporating a quenching step into the conjugate synthesis reaction, the inventors have addressed a problem concerning shelf-life stability of chelator-protein conjugates, (e.g., those produced by acylation reactions on protein amino groups).
Previously, others have used quenching steps in synthesis reactions, for example, to treat peptide-methotrexate conjugates. See Endo et al, U.S. Pat. No. 5,106,955; see also Brinkley, Bioconjugate Chem. 1992, Vol. 3, No. 1, pp. 1-13. However, quenching steps have not been previously used to treat 1,4,7,10-tetraaazacyclododecane N,N′,N″,N′″-tetraaacetic acid (DOTA)-peptide conjugates or derivatives thereof. See Lewis et al., Bioconjugate Chem. 1994, 5, 565-576; Govindan et al., Bioconjugate Chem., Vol. 9, No. 6, 1998, 773-782; Min et al., Bioconjugate Chem. Vol. 5, No. 2, 1994,101-104; U.S. Pat. No. 5,082,930; U.S. Pat. No. 5,435,990; U.S. Pat. No. 5,739,323; and U.S. Pat. No. 5,756,065 .
Most methods of synthesizing DOTA-peptide conjugates rely on formation of stable amide bonds between the DOTA molecule and one or more ε-amino groups on a lysine residue of the peptide. Although the previously described methods of forming DOTA-peptide conjugates may also result in the formation of unstable DOTA-ester bonds with the peptide, (e.g., at the hydroxyl group of a serine, threonine, or tyrosine), because DOTA-peptide synthesis reactions are typically performed at a high pH (>8.0), any unstable ester bonds would be expected to be readily hydrolyzed. Further, DOTA-peptide synthesis reactions typically result in a low substitution ratio of DOTA per peptide. For example, a synthesis reaction using a molar ratio of DOTA to peptide of ˜100:1 typically results in a substitution ratio of less than four (4) DOTA molecules per peptide in the synthesized conjugate. See Lewis et al., Bioconjugate Chem. 1994, 5, 565-576; and Govindan et al., Bioconjugate Chem., Vol. 9, No. 6, 1998; see also Griffiths et al., J. Nucl. Med. Vol. 44, No. 1, January 2003, 77-84. The low substitution ratio would suggest that the synthesized DOTA-peptide conjugate contains few unstable bonds (e.g., serine, threonine, and/or tyrosine esters) relative to stable bonds (e.g., lysine amides). As such, it would not be expected that the incorporation of a quenching step would significantly improve the stability and hence, the labeling efficiency, of DOTA-peptide conjugates stored over a period of time.